Paradigm of coinfection with viral and bacterial agents causing bovine respiratory disease complex (BRDC) in Central Serbia

Viral and bacterial agents causing bovine respiratory disease complex (BRDC) were examined over a five-year period (2015-2019). Thirty-eight farms of beef and dairy cattle from Central Serbia which showed symptoms of BRDC were observed. A total of 118 nasal swab specimens collected from diseased bullocks (n=55), cows (n=18) and calves (n=45) were chosen for the isolation of bacteria and identification by aerobic cultivation. The most commonly isolated bacterial pathogen was Pasteurella multocida (72.88% positive samples). Disk-diffusion method (antibiogram) showed that Amoxiclav and Enrofloxacin were the most efficient antibiotics against Pasteurella multocida isolates (66.67% and 54.17%, respectively). From all examined samples, using the Real Time RT-PCR and PCR methods, we determined the genome sequences of bovine respiratory syncytial virus (BRSV-34.78% of tested samples (8/23), bovine viral diarrhea virus (BVDV, in 34.37% (11/32) and Mycoplasma spp. in 33.33% (8/24). The genome of BoHV-1 virus was not detected in any of the 19 samples tested by Real Time PCR method.Publishe

Bioinformatics analysis of eukaryotic positively oriented single stranded RNA viruses

Birbica, Faculty of Sciences, Departmologia Seent of Biology and Ecology Positively oriented single stranded RNA viruses [ssRNA(+)] persistently affect health and well-being of all eukaryotes, including plants, animals and humans (i.e. SARS-CoV-2, yellow fever, hepatitis C, zika, West Nile, pepper mild mottle virus, etc.). How come these viruses are so wide spread and hard to eradicate? Besides their high changeability, another major reason is their ability to mimic host processes upon entering the host. Only recently it was revealed that ssRNA(+) viruses undergo methylation inside the host in the process that is similar to the methylation of the hosts’ own mRNAs. Such process may enable or disable virus to avoid some of the host’s defense mechanisms, but it inevitably impacts viral stability and fitness. Studies on this topic have only started, opening even more questions, with major ones being: how ssRNA(+) methylation, that occurs in the host, impacts viral pathogenicity and are these methylation patterns different in different hosts and for different ssRNA(+) viruses or do these viral methylomes share more universal pattern in concordance with their similar genome organization? Among numerous different methylation patterns of RNA, this research focused on N6-methyladenosine (m6A), as the most common and abundant methylation in eukaryotes, which was confirmed to be present in ssRNA(+) viruses as well. This study searched for patterns in the primary sequences and secondary structures of ssRNA(+) that are associated to m6A methylation sites relying on the experimentally obtained m6A datasets for eukaryotes and eukaryotic ssRNA(+) viruses. The results are discussed in view of datasets characteristics and study approach

Bioinformatics analysis of eukaryotic positively oriented single stranded RNA viruses

Positively oriented single stranded RNA viruses [ssRNA(+)] persistently affect health and well-being of all eukaryotes, including plants, animals and humans (i.e. SARS-CoV-2, yellow fever, hepatitis C, zika, West Nile, pepper mild mottle virus, etc.). How come these viruses are so wide spread and hard to eradicate? Besides their high changeability, another major reason is their ability to mimic host processes upon entering the host. Only recently it was revealed that ssRNA(+) viruses undergo methylation inside the host in the process that is similar to the methylation of the hosts’ own mRNAs. Such process may enable or disable virus to avoid some of the host’s defense mechanisms, but it inevitably impacts viral stability and fitness. Studies on this topic have only started, opening even more questions, with major ones being: how ssRNA(+) methylation, that occurs in the host, impacts viral pathogenicity and are these methylation patterns different in different hosts and for different ssRNA(+) viruses or do these viral methylomes share more universal pattern in concordance with their similar genome organization? Among numerous different methylation patterns of RNA, this research focused on N6-methyladenosine (m6A), as the most common and abundant methylation in eukaryotes, which was confirmed to be present in ssRNA(+) viruses as well. This study searched for patterns in the primary sequences and secondary structures of ssRNA(+) that are associated to m6A methylation sites relying on the experimentally obtained m6A datasets for eukaryotes and eukaryotic ssRNA(+) viruses. The results are discussed in view of datasets characteristics and study approach.Book of Abstracts: Belgrade BioInformatics Conference 202

Genome sequence diversity of SARS-CoV-2 in Serbia: insights gained from a 3-year pandemic study

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, has been evolving rapidly causing emergence of new variants and health uncertainties. Monitoring the evolution of the virus was of the utmost importance for public health interventions and the development of national and global mitigation strategies. Here, we report national data on the emergence of new variants, their distribution, and dynamics in a 3-year study conducted from March 2020 to the end of January 2023 in the Republic of Serbia. Nasopharyngeal and oropharyngeal swabs from 2,398 COVID-19-positive patients were collected and sequenced using three different next generation technologies: Oxford Nanopore, Ion Torrent, and DNBSeq. In the subset of 2,107 SARS-CoV-2 sequences which met the quality requirements, detection of mutations, assignment to SARS-CoV-2 lineages, and phylogenetic analysis were performed. During the 3-year period, we detected three variants of concern, namely, Alpha (5.6%), Delta (7.4%), and Omicron (70.3%) and one variant of interest—Omicron recombinant “Kraken” (XBB1.5) (<1%), whereas 16.8% of the samples belonged to other SARS-CoV-2 (sub)lineages. The detected SARS-CoV-2 (sub)lineages resulted in eight COVID-19 pandemic waves in Serbia, which correspond to the pandemic waves reported in Europe and the United States. Wave dynamics in Serbia showed the most resemblance with the profile of pandemic waves in southern Europe, consistent with the southeastern European location of Serbia. The samples were assigned to sixteen SARS-CoV-2 Nextstrain clades: 20A, 20B, 20C, 20D, 20E, 20G, 20I, 21J, 21K, 21L, 22A, 22B, 22C, 22D, 22E, and 22F and six different Omicron recombinants (XZ, XAZ, XAS, XBB, XBF, and XBK). The 10 most common mutations detected in the coding and untranslated regions of the SARS-CoV-2 genomes included four mutations affecting the spike protein (S:D614G, S:T478K, S:P681H, and S:S477N) and one mutation at each of the following positions: 5′-untranslated region (5’UTR:241); N protein (N:RG203KR); NSP3 protein (NSP3:F106F); NSP4 protein (NSP4:T492I); NSP6 protein (NSP6: S106/G107/F108 - triple deletion), and NSP12b protein (NSP12b:P314L). This national-level study is the most comprehensive in terms of sequencing and genomic surveillance of SARS-CoV-2 during the pandemic in Serbia, highlighting the importance of establishing and maintaining good national practice for monitoring SARS-CoV-2 and other viruses circulating worldwide.The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, has been evolving rapidly causing emergence of new variants and health uncertainties. Monitoring the evolution of the virus was of the utmost importance for public health interventions and the development of national and global mitigation strategies. Here, we report national data on the emergence of new variants, their distribution, and dynamics in a 3-year study conducted from March 2020 to the end of January 2023 in the Republic of Serbia. Nasopharyngeal and oropharyngeal swabs from 2,398 COVID-19- positive patients were collected and sequenced using three different next generation technologies: Oxford Nanopore, Ion Torrent, and DNBSeq. In the subset of 2,107 SARS-CoV-2 sequences which met the quality requirements, detection of mutations, assignment to SARS-CoV-2 lineages, and phylogenetic analysis were performed. During the 3-year period, we detected three variants of concern, namely, Alpha (5.6%), Delta (7.4%), and Omicron (70.3%) and one variant of interest—Omicron recombinant “Kraken” (XBB1.5) (<1%), whereas 16.8% of the samples belonged to other SARS-CoV-2 (sub)lineages. The detected SARS-CoV-2 (sub)lineages resulted in eight COVID-19 pandemic waves in Serbia, which correspond to the pandemic waves reported in Europe and the United States. Wave dynamics in Serbia showed the most resemblance with the profile of pandemic waves in southern Europe, consistent with the southeastern European location of Serbia. The samples were assigned to sixteen SARS-CoV-2 Nextstrain clades: 20A, 20B, 20C, 20D, 20E, 20G, 20I, 21J, 21K, 21L, 22A, 22B, 22C, 22D, 22E, and 22F and six different Omicron recombinants (XZ, XAZ, XAS, XBB, XBF, and XBK). The 10 most common mutations detected in the coding and untranslated regions of the SARS-CoV-2 genomes included four mutations affecting the spike protein (S:D614G, S:T478K, S:P681H, and S:S477N) and one mutation at each of the following positions: 5′-untranslated region (5’UTR:241); N protein (N:RG203KR); NSP3 protein (NSP3:F106F); NSP4 protein (NSP4:T492I); NSP6 protein (NSP6: S106/G107/F108 - triple deletion), and NSP12b protein (NSP12b:P314L). This national-level study is the most comprehensive in terms of sequencing and genomic surveillance of SARS-CoV-2 during the pandemic in Serbia, highlighting the importance of establishing and maintaining good national practice for monitoring SARS-CoV-2 and other viruses circulating worldwide

Genetska analiza i distribucija parvovirusa (ppvs) detektovanih u organima divljih svinja u Srbiji

Porcine parvoviruses (PPVs) are diverse and persistently evolving viruses found in domestic pigs and wild boars. Porcine parvovirus 1 (PPV1) causes reproductive problems in adult animals, although the veterinary relevance of PPV2, PPV3, and PPV4 has not been clarified. The detection and sequence analysis of PPVs circulating in wild boar populations in Serbia was performed to determine their phylogenetic relationships and prevalence in 122 organ samples collected during 2018. The DNA of PPV1, PPV2, and PPV3 was detected in 56.6% of the examined samples, whilst PPV4 was not identified. Overall, PPV3 was the most prevalent in 69.6% of the positive samples, followed by PPV1 in 63.8%, and PPV2 in 21.7% samples. Single infections were more common, although concurrent infections were confirmed in 34.8% samples for two, and 10.1% samples for three viruses. Sequence analysis of wild boar PPV1 showed no significant nucleotide differences from domestic pig PPV1 strains detected in Europe and the USA, however separate clustering from strains from China and the NADL-2 strain was demonstrated. Examination of the selected PPV2 sequences might suggest a certain geographical distribution of genetically diverse PPV2 strains considering high similarities to the strains from neighboring countries, and variability in comparison with other reported PPV2 sequences from different parts of the world. Wild boar PPV3 sequences clustered separately from most of the strains detected in wild boars, as well as the original porcine hokovirus strain. It is further noted that genetically different PPV3 strains circulate amongst Serbian domestic pigs and wild boars.Parvovirusi svinja predstavljaju genetski različite viruse koji izazivaju infekcije doma-ćih i divljih svinja. Parvovirus svinja 1 (PPV1) dovodi do pojave reproduktivnih pro-blema kod odraslih jedinki, dok klinički značaj PPV2, PPV3 i PPV4 još uvek nije u potpunosti razjašnjen. Izvršena je detekcija i analiza genetskih sekvenci parvovirusa koji cirkulišu u populaciji divljih svinja u Srbiji u cilju njihove fi logenetske analize i određivanja zastupljenosti u ukupno 122 uzorka organa prikupljenih tokom 2018. go-dine. Prisustvo DNK PPV1, PPV2 i PPV3 detektovano je u 56,6% ispitanih uzoraka, pri čemu prisustvo PPV4 nije utvrđeno. Među pozitivnim uzorcima, PPV3 je pro-centualno najzastupljeniji virus detektovan u 69,6%, dok je prisustvo PPV1 i PPV2 utvrđeno u 63,8%, odnosno u 21,7% pozitivnih uzoraka. Infekcije jednim virusom su češće identifi kovane, međutim, prisustvo mešovitih infekcija sa dva, odnosno tri par-vovirusa zabeleženo je u 34,8% i 10,1% uzoraka. Analizom genetskih sekvenci PPV1 detektovanih kod divljih svinja nisu utvrđene značajnije razlike u odnosu na analogne sekvence PPV1 poreklom od domaćih svinja iz Evrope i SAD, međutim zabeleženo je izdvajanje u zaseban klaster u odnosu na kineske sojeve virusa i soj NADL-2. Ispi-tivanjem sekvenci PPV2 utvrđena je izvesna geografska distribucija genetski različitih sojeva navedenog virusa s obzirom na njihovu veliku sličnost sa sojevima virusa iz su-sednih zemalja. Sekvence PPV3 detektovanih kod divljih svinja su se na fi logenetskom stablu izdvajale u zaseban klaster u odnosu na većinu dostupnih sekvenci navedenog virusa detektovanih kod divljih svinja. Pored toga, zabeleženo je da genetski različiti sojevi PPV3 cirkulišu u populacijama divljih i domaćih svinja u Srbiji

Importance of multidisciplinary and regional collaboration in integrated West Nile virus surveillance - the "One Health" concept

West Nile virus (WNV) danas je jedan od najrasprostranjenijih arbovirusa. U posljednja se dva desetljeća WNV infekcije kontinuirano pojavljuju na području Europe u obliku manjih ili većih epidemija i epizootija. U ovom preglednom radu prikazujemo epidemiološke značajke WNV infekcija koje su dokazane na području Hrvatske i Srbije u kontekstu “Jednog zdravlja”. Prikazani rezultati potvrđuju značaj multidisciplinarne i regionalne suradnje u dijagnostici i praćenju ove (re-)emergentne virusne zoonoze.West Nile virus (WNV) is nowadays one of the most widely distributed arboviruses. In the last two decades, small or large WNV epidemics and epizootics are continuously reported in Europe. In this review, we present the epidemiological characteristics of WNV infections detected in Croatia and Serbia within the "One Health" context. Presented results confirm the importance of multidisciplinary and regional collaboration in the diagnosis and surveillance of this (re-)emerging viral zoonosis

Kvalitet života osoba oštećenog sluha

Specifičnosti oštećenja sluha – Novi korac

Development of multiplex PCR based NGS protocol for whole genome sequencing of West Nile virus lineage 2 directly from biological samples using Oxford Nanopore platform

West Nile virus (WNV) can affect humans, birds, horses and another mammals, causing asymptomatic infection, mild febrile disease, neurological and systematic disease and death. In order to gain insight into the prevalence of WNV, a monitoring program has been established in the Republic of Serbia. Whole genome sequencing is essential for the molecular epizootiological analysis of virus entry and transmission routes, especially in high-risk regions. This paper describes the development of a multiplex PCR based NGS protocol for whole genome sequencing of WNV lineage 2 directly from biological samples using Oxford Nanopore (ONT) platform. The results obtained using this platform, confirmed by Sanger sequencing, indicate that this protocol can be applied to obtain whole sequences of the WNV genome, even when the virus concentration in the sample is medium, Ct value is approximately 30. The use of this protocol does not require prior virus isolation on cell culture nor the depletion of host nucleic acids